Daily rhythms and behavior are driven in part by biological clocks. Clinical studies suggest that circadian rhythm defects contribute to certain forms of depression and sleep disorders. Much of the understanding of molecular clock mechanisms is derived from genetic studies in the fruit fly, Drosophila melanogaster. Analysis of circadian rhythm genes reveal that transcriptional feedback loops are at the core of circadian pacemakers. The mammalian counterparts of fly genes are also intimately involved in circadian clocks, establishing the fly as an important model system for clock gene discovery. Protein phosphorylation is increasingly being appreciated as important for the timing of biological clocks. Interestingly, a mutation in the human ortholog of the fly rhythm gene period leads to a sleep disorder in which the mutant protein is a defective substrate for phosphorylation. Recent work has identified a novel role for the protein kinase, casein kinase 2 (CK2), in the Drosophila clock. CK2 can directly phosphorylate the circadian rhythm proteins, PERIOD (PER) and TIMELESS (TIM) in vitro and PER and TIM protein disappearance is delayed in CK2 mutants, implicating this kinase in regulating PER and TIM stability. To determine if CK2 alpha is required for circadian function, genetic screens will be performed to identify null alleles of CK2 alpha. Such screens may also identify modifiers of CK2 function. The circadian regulation of CK2 alpha subcellular localization will be investigated. To address the in vivo role of CK2 alpha in PER and TIM phosphorylation, phosphorylation state as well as stability will be examined in homozygous CK2 alpha mutants. Coimmunoprecipitation studies and searches for in vitro and in vivo phosphorylation sites will be performed to establish direct functional interactions. Surprisingly, CK2 alpha is also co-expressed with the key circadian neuropeptide PIGMENT DISPERSING FACTOR (PDF) in the axons and termini of pacemaker neurons, suggesting a novel link between the central pacemaker and neuronal output. The role of CK2 in regulating various aspects of PDF expression and amidation will be addressed. In addition, the association of CK2 with PDF will be examined using electron microscopy. The discovery of a circadian function of CK2 in plants, animals, and fungi suggests these studies will be relevant to many biological systems, including humans.